Fig 2: In silico screening approach.(A) Schematic representation of TSLP, TSLPR and IL-7Rα forming a ternary complex; binding interfaces are indicated as site I, II, and III. (B) Overview of the virtual screening of potential small-molecule TSLP inhibitors and compound selection. A library of compounds from Specs and VitasM was screened using 3D pharmacophore models. (C, D) In vitro screening of twelve potential hit compounds in TSLP-activated HuT78 cells. Cells were treated with different concentrations of putative TSLP/TSLP receptor inhibitors for 36 h. IL-4 and IL-13 expression were detected with ELISA (n = 3). (E, F) Inhibition of Th2 cytokine secretion from human primary CD4 + T cells. TSLP-activated human primary CD4 + T cells from three donors were treated with different concentrations of compound 7, 13, and 14 for 36 h followed by quantification of IL-13 and IL-4 secretion via ELISA (n = 3). IC50 values [μM] and slope were determined using GraphPad Prism software. Data information: All data are presented as mean ± SEM. n represents the number of biological replicates. Statistical analysis was performed using Student’s t test, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. Source data are available online for this figure.

Fig 3: Development & utilization of an atopic diseases-organ chip for preclinical BP79 testing.(A, B) Experimental setup of the co-cultivation of normal or atopic-like skin models with 3D normal bronchial epithelial models in a microfluidic two-organ chip. BP79 and tacrolimus were topically applied onto the skin (disease) model. (C) H&E staining of co-cultivated skin (normal and diseased) and lung tissue models w/ or w/o topical application of BP79 or tacrolimus. (D) Topical application of BP79 significantly reduced IL-13, IL-4, TSLP and periostin expression as detected via ELISA. Scale bar = 50 μm. (E) Relative mRNA expression of TSLP, filaggrin, keratin 5, keratin 14, and keratin 10 in atopic-like skin and bronchial lung tissue models following topical treatment with BP79 and tacrolimus determined via quantitative RT-PCR (n = 3). GAPDH served as a housekeeping gene.

Fig 4: Target engagement and specificity of BP79.(A) Proximity ligation assay (PLA): TSLP binds to TSLPR and forms a ternary complex with IL-7Rα at the cell surface initiating intracellular signaling. PLA was performed to verify the inhibition of TSLP-mediated ternary complex formation via BP79. Primary keratinocyte cells were treated with the TSLP inhibitors and stimulated with TSLP. Cells were fixed and the interaction between TSLPR and IL-7Rα was observed with PLA assay. BP79 inhibited the TSLP:TSLPR:IL-7Rα ternary complex formation. Images were counted using ImageJ (n = 20). Scale bar = 50 μm. (B) In vitro thermal shift assay. BP79 thermally stabilized TSLPR up to 45 °C. (C) To determine if BP79 decreases the cytokine expression through direct binding of upstream kinases such as JAK1/2 or other kinase targets, BP79 was screened against 97 kinase targets using the KINOMEscan™ platform (radioligand displacement assay performed by Eurofins Discovery Services). Data information: All data are presented as mean ± SEM. n represents the number of biological replicates. Statistical analysis was performed using Student’s t test, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. Source data are available online for this figure.

Fig 5: Functional validation of BP79.(A, B) TSLP-activated primary CD4 + T cells were treated with different concentrations of the TSLP inhibitors BP75 and BP79 for 36 h, respectively. IL-13 and IL-4 secretion was quantified by ELISA (n = 6), mean ± SEM. IC50 values [μM] and slope were determined using GraphPad Prism software. (C–E) Cytotoxicity of BP79 in primary human keratinocytes (n = 4), fibroblasts (n = 5), and CD4 + T cells (n = 5) was determined by MTT assay. (F) Inhibition of TSLP-mediated T-cell hyperproliferation. Primary CD4 + T cells were treated with TSLP inhibitors (or DMSO) and stimulated with TSLP for 1, 2 and 5 days. Cell proliferation was determined using a cell counter; n = 3. (G) Primary human CD4 + T cells and (H) keratinocytes were treated with BP79 and stimulated with TSLP. BP79 inhibited STAT6 and STAT3 activation in primary CD4 + T cells and primary keratinocytes, respectively (n = 3). The band intensities were quantified using ImageJ (n = 3). Phospho-Stat6 and Phospho-Stat3 expressions were normalized to Stat6 and Stat3 expressions, respectively. (I) Primary human myeloid dendritic cells were treated with BP79 and stimulated with TSLP for 24 h. OX-40L expression was detected using flow cytometry. (J) Primary human myeloid dendritic cells from three donors were treated with BP79 and stimulated with TSLP for 24 h. CCL17 expression was detected using ELISA (n = 3). (K) Inhibition of dendritic cell-mediated Th2 cell differentiation. IL-13 secretion was determined using ELISA (n = 3). Data information: All data are presented as mean ± SEM. n represents the number of biological replicates. Statistical analysis was performed using Student’s t test, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. Source data are available online for this figure.